Ampa-binding human GluR3 receptors

ABSTRACT

Described herein are isolated polynucleotides which code for a family of AMPA-type human CNS receptors. The receptors are characterized structurally and the construction and use of cell lines expressing these receptors are disclosed.

This application is a division, of application Ser. No. 07/896,612,filed Jun. 10, 1992, now abandoned.

FIELD OF THE INVENTION

This invention is concerned with applications of recombinant DNAtechnology in the field of neurobiology. More particularly, theinvention relates to the cloning and expression of DNA coding forexcitatory amino acid (EAA) receptors, especially human EAA receptors.

BACKGROUND TO THE INVENTION

In the mammalian central nervous system (CNS), the transmission of nerveimpluses is controlled by the interaction between a neurotransmittersubstance released by the “sending” neuron which then binds to a surfacereceptor on the “receiving” neuron to cause excitation thereof.L-glutamate is the most abundant neurotransmitter in the CNS, andmediates the major excitatory pathway in vertebrates. Glutamate istherefore referred to as an excitatory amino acid (EAA) and thereceptors which respond to it are variously referred to as glutamatereceptors, or more commonly as EAA receptors.

Using tissues isolated from mammalian brain, and various synthetic EAAreceptor agonists, knowledge of EAA receptor pharmacology has beenrefined somewhat. Members of the EAA receptor family are now groupedinto three main types based on differential binding to such agonists.One type of EAA receptor, which in addition to glutamate also binds theagonist NMDA (N-methyl-D-aspartate), is referred to as the NMDA type ofEAA receptor. Two other glutamate-binding types of EAA receptor, whichdo not bind NMDA, are named according to their preference for bindingwith two other EAA receptor agonists, namely AMPA(alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate.Particularly, receptors which bind glutamate but not NMDA, and whichbind with greater affinity to kainate than to AMPA, are referred to askainate type EAA receptors. Similarly, those EAA receptors which bindglutamate but not NMDA, and which bind AMPA with greater affinity thankainate are referred to as AMPA type EAA receptors.

The glutamate-binding EAA receptor family is of great physiological andmedical importance. Glutamate is involved in many aspects of long-termpotentiation (learning and memory), in the development of synapticplasticity, in epileptic seizures, in neuronal damage caused by ischemiafollowing stroke or other hypoxic events, as well as in other forms ofneurodegenerative processes. However, the development of therapeuticswhich modulate these processes has been very difficult, due to the lackof any homogeneous source of receptor material with which to discoverselectively binding drug molecules, which interact specifically at theinterface of the EAA receptor. The brain derived tissues currently usedto screen candidate drugs are heterogeneous receptor sources, possessingon their surface many receptor types which interfere with studies of theEAA receptor/ligand interface of interest. The search for humantherapeutics is further complicated by the limited availability of braintissue of human origin. It would therefore be desirable to obtain cellsthat are genetically engineered to produce only the receptor ofinterest. With cell lines expressing cloned receptor genes, a substratewhich is homogeneous for the desired receptor is provided, for drugscreening programs.

Very recently, genes encoding substituent polypeptides of EAA receptor sfrom non-human sources, principally rat, have been discovered. Hollmannet al., Nature 342: 643, 1989 described the isolation from rat of a genereferred to originally as GluR-K1 (but now called simply GluR1). Thisgene encodes a member of the rat EAA receptor family, and was originallysuspected as being of the kainate type. Subsequent studies by Keinanenet al., Science 249: 556, 1990, showed, again in rat, that a gene calledGluR-A, which was in fact identical to the previously isolated GluR1, infact encodes a receptor not of the kainate type, but rather of the AMPAtype. These two groups of researchers have since reported as many asfive related genes isolated from rat sources. Boulter et al., Science249: 1033, 1990, revealed that, in addition to GluR1, the rat contained3 other related genes, which they called GluR2, GluR3, and GluR4, andBettler et al., Neuron 5: 583. 1990 described GluR5. Keinanen et al.,supra, described genes called GluR-A, GluR-B, GluR-C and GluR-D whichcorrespond precisely to GluR1, GluR2, GluR3 and GluR4 respectively.Sommer et al., Science 249: 1580, 1990 also showed, for GluR-A, GluR-B,GluR-C and GluR-D two alternatively spliced forms for each gene. Theseauthors, as well as Monyer et al., Neuron 6: 799, 1991 were able to showthat the differently spliced versions of these genes were differentiallyexpressed in the rat brain. In addition to the isolation of these AMPAreceptor genes, several studies have more recently attempted todetermine the ion-gating properties of different mixtures of the knownreceptors (Nakanishi et al., Neuron 5: 569, 1990; Hollmann et a.,Science 252: 851, 1991; Verdoorn et al., Science 252: 1715, 1991; andsee WO 91/06648).

There has emerged from these molecular cloning advances a betterunderstanding of the structural features of EAA receptors and theirsubunits, as they exist in the rat brain. According to the current modelof EAA receptor structure, each is heteromeric in structure, consistingof individual membrane-anchored subunits, each having four transmembraneregions, and extracellular domains that dictate ligand bindingproperties to some extent and contribute to the ion-gating functionserved by the receptor complex. Keinanen et al, supra, have shown forexample that each subunit of the rat GluR receptor, including thosedesignated GluR-A, GluR-B, GluR-C and GluR-D, display cation channelactivity gated by glutamate, by AMPA and by kainate, in their unitarystate. When expressed in combination however, for example GluR-A incombination with GluR-B, gated ion channels with notably larger currentsare produced by the host mammalian cells.

In the search for therapeutics useful to treat CNS disorders in humans,it is highly desirable of course to provide a screen for candidatecompounds that is more representative of the human situation than ispossible with the rat receptors isolated to date. It is particularlydesirable to provide cloned genes coding for human receptors, and celllines expressing those genes, in order to generate a proper screen forhuman therapeutic compounds. These, accordingly, are objects of thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides a family of isolated polynucleotides thatcode for AMPA-binding human EAA receptors. By providing polynucleotidesthat code specifically for CNS receptors native to humans, the presentinvention provides means for evaluating the human nervous system, andparticularly for assessing potentially therapeutic interactions betweenthe AMPA-binding human EAA receptors and selected natural and syntheticligands.

In one of its aspects, the present invention provides an isolatedpolynucleotide comprising nucleic acids arranged in a sequence thatcodes for an EAA receptor belonging to the human GluR3 family.Alternatively, the polynucleotide may code for an AMPA-binding fragmentof a human GluR3 receptor, or for an AMPA-binding variant of a humanGluR3 receptor. According to one embodiment of the present invention,the isolated polynucleotide encodes a receptor comprising amino acids,arranged in the sequence herein specified with reference to FIGS. 1A-1E(SEQ ID NOS: 1 and 2) and referred to as the human GluR3A receptor.According to another embodiment of the invention, the polynucleotideencodes a variant of the human GluR3A receptor, which variant has theamino acid sequence herein specified with reference to FIGS. 3A-3F (SEQID NOS: 3 and 4) and is herein referred to as the human GluR3B receptor.In various specific embodiments of the present invention, thepolynucleotide consists of DNA e.g. cDNA, or of RNA e.g. messenger RNA.In other embodiments of the present invention, the polynucleotide may becoupled to a reporter molecule, such as a radioactive label, for use inautoradiographic studies of human GluR3 receptor tissue distribution. Infurther embodiments of the present invention, fragments of thepolynucleotides of the invention, including radiolabelled versionsthereof, may be employed either as probes for detection of glutamatereceptor-encoding polynucleotides, as primers appropriate for amplifyingsuch polynucleotides present in a biological specimen, or as templatesfor expression of a GluR3 receptor or an AMPA-binding fragments orvariant thereof.

According to another aspect of the present invention, there is provideda cellular host having incorporated therein a polynucleotide of thepresent invention. In embodiments of the present invention, thepolynucleotide is a DNA molecule and is incorporated for expression andsecretion in the cellular host, to yield a functional, membrane-boundhuman GluR3 receptor. In other embodiments of the present invention, thepolynucleotide is an RNA molecule which is incorporated in the cellularhost to yield the human GluR3 receptor as a functional, membrane-boundproduct of translation.

According to another aspect of the invention, there is provided aprocess for obtaining a substantially homogeneous source of a human EAAreceptor useful for performing ligand binding assays, which comprisesthe steps of culturing a genetically engineered cellular host of theinvention, and then recovering the cultured cells. Optionally, thecultured cells may be treated to obtain membrane preparations thereof,for use in the ligand binding assays.

According to another aspect of the present invention, there is provideda method for assaying interaction between a test ligand and a human EAAreceptor, comprising the steps of incubating the test ligand underappropriate conditions with a human GluR3 receptor source, i.e., acellular host of the invention or a membrane preparation derivedtherefrom, and then determining between the substance and the receptorsource.

These and other aspects of the invention are now described in greaterdetail with reference to the accompanying drawings, in which:

BRIEF REFERENCE TO THE DRAWINGS

FIGS. 1A-1E provide a DNA sequence (SEQ ID NO: 1) coding for the humanGluR3A receptor, and the amino acid (SEQ ID NO: 2) sequence thereof;

FIG. 2 depicts the strategy employed in cloning the human GluR3Areceptor-encoding DNA illustrated in FIGS. 1A-1E;

FIGS. 3A-3F provide a DNA sequence (SEQ ID NO: 3) coding for the humanGluR3B receptor, and the amino acid (SEQ ID NO: 4) sequence thereof;

FIG. 4 depicts the strategy employed in cloning the human GluR3Breceptor-encoding DNA illustrated in FIGS. 3A-3F;

FIG. 5 depicts the strategy employed in generating recombinant DNAexpression constructs incorporating the receptor-encoding DNA;

FIG. 6 provides the amino acid sequence of the human GluR3A receptor(SEQ ID NO: 5) and the human GluR3B receptor (SEQ ID NO: 7) in a regionof dissimilarity; and

FIG. 7 illustrates the AMPA-binding property of the human GluR3Areceptor.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

The invention relates to human CNS receptors of the AMPA-binding type,and provides isolated polynucleotides that code for such receptors. Theterm “isolated” is used herein with reference to intact polynucleotidesthat are generally less than about 4,000 nucleotides in length and whichare otherwise isolated from DNA coding for other human proteins.

In the present context, human CNS receptors of the AMPA-binding typeexhibit a characteristic ligand binding profile, which reveals glutamatebinding and relative greater affinity for binding AMPA than for otherbinding other CNS receptor ligands such as kainate, glutamate and theirclosely related analogues.

In the present specification, an AMPA-binding receptor is said to be“functional” if a cellular host producing it exhibits de novo channelactivity when exposed appropriately to AMPA, as determined by theestablished electrophysiological assays described for example by Hollmanet al, supra, or by any other assay appropriate for detectingconductance across a cell membrane.

Members of the human GluR3 family of the invention possess structuralfeatures characteristic of the EAA receptors in general, includingextracellular N- and C-terminal regions, as well as four internalhydrophobic domains which serve to anchor the receptor within the cellsurface membrane. The GluR3A member of the human GluR3 family is aprotein characterized structurally as a single polypeptide chain that isproduced initially in precursor form bearing a 22 amino acid residueN-terminal signal peptide, and is transported to the cell surface inmature form, lacking the signal peptide and consisting of 866 aminoacids arranged in the sequence illustrated, by single letter code, inFIGS. 1A-1E and SEQ ID NO: 2. Unless otherwise stated, the term humanGluR3A receptor refers to the mature form of the receptor, and aminoacid residues of the individual human GluR3 receptors are accordinglynumbered with reference to the mature protein sequence. With respect tostructural domains of the receptor, hydropathy analysis reveals fourputative transmembrane domains, one spanning residues 527-546 inclusive(TM-1), another spanning residues 575-593 (TM-2), a third spanningresidues 604-622 (TM-3) and the fourth spanning residues 796-816 (TM-4).Based on this assignment, it is likely that the human GluR3A receptorstructure, in its natural membrane-bound form, consists of a 526 aminoacid N-terminal extracellular domain, followed by a hydrophobic regioncontaining four transmembrane domains and an extracellular, 50 aminoacid C-terminal domain.

As shown in FIGS. 3A-3F and SEQ ID NOS: 3 and 4, a structurally relatedvariant of the human GluR3A receptor that occurs naturally in humanbrain tissue has also been identified, and is designated herein as thehuman GluR3B receptor. Like GluR3A, the GluR3B receptor is also 866amino acids in length in its mature, membrane-bound form, and initiallybears a signal peptide identical to that borne on the GluR3A receptor.Four transmembrane domains are also apparent from the GluR3B sequence,and indicate that these domains lie in the same amino acid regions justindicated in connection with the GluR3A receptor.

With respect to primary structure, the human GluR3B receptor differsfrom the GluR3A receptor in a 36 amino acid region separatingtransmembrane domains TM-3 and TM-4, i.e. residues 748-783. Forcomparison, the sequences of GluR3A and GluR3B in this region arecompared in FIG. 6 and are also show in SEQ ID NOS: 5 and 6.

Binding assays performed with various ligands, and with membranepreparations derived from mammalian cells engineered genetically toproduce the human GluR3 receptors in membrane-bound form indicate thatboth human GluR3A and human GluR3B bind selectively to AMPA, relativeparticularly to kainate and NMDA. This feature, coupled with themedically significant connection between AMPA-type receptors andneurological disorders and disease indicate that the present receptors,as well as AMPA-binding fragments and variants thereof, will serve asvaluable tools in the screening and discovery of ligands useful tomodulate in vivo interactions between such receptors and their naturalligand, glutamate. Thus, a key aspect of the present invention residesin the construction of cells that are engineered genetically to producehuman GluR3 receptor, to serve as a ready and homogeneous source ofreceptor for use in in vitro ligand binding and/or channel activationassays.

For use in the ligand binding assays, it is desirable to construct byapplication of genetic engineering techniques a mammalian cell thatproduces a human GluR3 receptor as a heterologous and membrane-boundproduct. According to one embodiment of the invention, the constructionof such engineered cells is achieved by introducing into a selected hostcell a recombinant DNA construct in which DNA coding for a secretableform of the desired human GluR3 receptor, i.e., a form bearing itsnative signal peptide or a functional, heterologous equivalent thereof,is linked operably with expression controlling elements that arefunctional in the selected host to drive expression of thereceptor-encoding DNA, and thus elaborate the desired human GluR3receptor protein. Such cells are herein characterized as having thereceptor-encoding DNA incorporated “expressibly” therein. Thereceptor-encoding DNA is referred to as “heterologous” with respect tothe particular cellular host if such DNA is not naturally found in theparticular host. The particular cell type selected to serve as host forproduction of the human GluR3 receptor can be any of several cell typescurrently available in the art, but should not of course be a cell typethat in its natural state elaborates a surface receptor that can bindexcitatory amino acids, and so confuse the assay results sought from theengineered cell line. Generally, such problems are avoided by selectingas host a non-neuronal cell type, and can further be avoided usingnon-human cell lines, as is conventional. It will be appreciated thatneuronal- and human-type cells may nevetheless serve as expressionhosts, provided that “background” binding to the test ligand isaccounted for in the assay results.

According to one embodiment of the present invention, the cell lineselected to serve as host for human GluR3 receptor production is amammalian cell. Several types of such cell lines are currently availablefor genetic engineering work, and these include the chinese hamsterovary (CHO) cells for example of K1 lineage (ATCC CCL 61) including thePro5 variant (ATCC CRL 1281); the fibroblast-like cells derived fromSV40-transformed African Green monkey kidney of the CV-1 lineage (ATCCCCL 70), of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage(ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658),murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCCCRL 1573), human carcinoma cells including those of the HeLa lineage(ATCC CCL 2), and neuroblastoma cells of the lines IMR-32 (ATCC CCL127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11).

A variety of gene expression systems have been adapted for use withthese hosts and are now commercially available, and any one of thesesystems can be selected to drive expression of the human GluR3receptor-encoding DNA. These systems, available typically in the form ofplasmidic vectors, incorporate expression cassettes the functionalcomponents of which include DNA constituting expression controllingsequences, which are host-recognized and enable expression of thereceptor-encoding DNA when linked 5′ thereof. The systems furtherincorporate DNA sequences which terminate expression when linked 3′ ofthe receptor-encoding region. Thus, for expression in the selectedmammalian cell host, there is generated a recombinant DNA expressionconstruct in which DNA coding for a secretable form of the receptor islinked with expression controlling DNA sequences recognized by the host,and which include a region 5′ of the receptor-encoding DNA to driveexpression, and a 3′ region to terminate expression. The plasmidicvector harbouring the recombinant DNA expression construct typicallyincorporates such other functional components as an origin ofreplication, usually virally-derived, to permit replication of theplasmid in the expression host and desirably also for plasmidamplification in a bacterial host, such as E. coli. To provide a markerenabling selection of stably transformed recombinant cells, the vectorwill also incorporate a gene conferring some survival advantage on thetransformants, such as a gene coding for neomycin resistance in whichcase the transformants are plated in medium supplemented with neomycin.

Included among the various recombinant DNA expression systems that canbe used to achieve mammalian cell expression of the receptor-encodingDNA are those that exploit promoters of viruses that infect mammaliancells, such as the promoter from the cytomegalovirus (CMV), the Roussarcoma virus (RSV), simian virus (SV40), murine mammary tumor virus(MMTV) and others. Also useful to drive expression are promoters such asthe LTR of retroviruses, insect cell promoters such as those regulatedby temperature, and isolated from Drosophila, as well as mammalian genepromoters such as those regulated by heavy metals i.e. themetalothionein gene promoter, and other steroid-inducible promoters.

For incorporation into the recombinant DNA expression vector, DNA codingfor a selected human GluR3 receptor, e.g. the human GluR3A receptor, thehuman GluR3B receptor or an AMPA-binding fragment or variant thereof,can be obtained by applying selected techniques of gene isolation orgene synthesis. As described in more detail in the examples herein, thehuman GluR3A receptor and the human GluR3B receptor are encoded withinthe genome of human brain tissue, and can therefore be obtained fromhuman DNA libraries by careful application of conventional geneisolation and cloning techniques. This typically will entail extractionof total messenger RNA from a fresh source of human brain tissue,preferably cerebellum or hippocampus tissue, followed by conversion ofmessage to cDNA and formation of a library in for example a bacterialplasmid, more typically a bacteriophage. Such bacteriophage harbouringfragments of the human DNA are typically grown by plating on a lawn ofsusceptible E. coli bacteria, such that individual phage plaques orcolonies can be isolated. The DNA carried by the phage colony is thentypically immobilized on a nitrocellulose or nylon-based hybridizationmembrane, and then hybridized, under carefully controlled conditions, toa radioactively (or otherwise) labelled oligonucleotide probe ofappropriate sequence to identify the particular phage colony carryingreceptor-encoding DNA or fragment thereof. Typically, the gene or aportion thereof so identified is subcloned into a plasmidic vector fornucleic acid sequence analysis.

In a specific embodiment of the invention, the selected GluR3 receptoris encoded by the DNA sequence illustrated In FIGS. 1A-1E SEQ ID NO: 1,for the GluR3A receptor, and by the DNA sequence illustrated in FIGS.3A-3F, SEQ ID NO: 3for the GluR3B receptor. In an obvious alternative,the DNA sequences coding for the selected receptor may be a synonymouscodon equivalent of the illustrated DNA sequences.

The illustrated DNA sequences constitute cDNA sequences identified inhuman brain cDNA libraries in the manner exemplified herein. Havingherein provided the nucleotide sequence of various members of the humanGluR3 receptor family, however, it will be appreciated thatpolynucleotides encoding the receptors can be obtained by other routes.Automated techniques of gene synthesis and/or amplification can beperformed to generate DNA coding therefor. Because of the length of thehuman GluR3 receptor-encoding DNA, application of automated synthesismay require staged gene construction, in which regions of the gene up toabout 300 nucleotides in length are synthesized individually and thenligated in correct succession by overhang complementarity for finalassembly. Individually synthesized gene regions can be amplified priorto assembly, using established polymerase chain reaction (PCR)technology.

The application of automated gene synthesis techniques provides anopportunity for generating polynucleotides that encode variants ofnaturally occurring human GluR3A and GluR3B receptors. It will beappreciated, for example, that polynucleotides coding for the humanGluR3 receptors herein described can be generated by substitutingsynonymous codons for those represented in the naturally occurringpolynucleotide sequences herein identified. In addition, polynucleotidescoding for human GluR3 receptor variants can be generated which forexample incorporate one or more e.g. 1-10, single amino acidsubstitutions, deletions or additions. Since it will for the most partbe desirable to retain the natural ligand binding profile of thereceptor for screening purposes, it is desirable to limit amino acidsubstitutions, for example to the so-called conservative replacements inwhich amino acids of like charge are substituted, and to limitsubstitutions to those sites less critical for receptor activity e.g.within about the first 20 N-terminal residues of the mature receptor,and such other regions as are elucidated upon receptor domain mapping.

With appropriate template DNA in hand, the technique of PCRamplification may also be used to directly generate all or part of thefinal gene. In this case, primers are synthesized which will prime thePCR amplification of the final product, either in one piece, or inseveral pieces that may be ligated together. This may be via step-wiseligation of blunt ended, amplified DNA fragments, or preferentially viastep-wise ligation of fragments containing naturally occurringrestriction endonuclease sites. In this application, it is possible touse either cDNA or genomic DNA as the template for the PCRamplification. In the former case, the cDNA template can be obtainedfrom commercially available or self-constructed cDNA libraries ofvarious human brain tissues, including hippocampus and cerebellum.

Once obtained, the receptor-encoding DNA is incorporated for expressioninto any suitable expression vector, and host cells are transfectedtherewith using conventional procedures, such as DNA-mediatedtransformation, electroporation, or particle gun transformation.Expression vectors may be selected to provide transformed cell linesthat express the receptor-encoding DNA either transiently or in a stablemanner. For transient expression, host cells are typically transformedwith an expression vector harbouring an origin of replication functionalin a mammalian cell. For stable expression, such replication origins areunnecessary, but the vectors will typically harbour a gene coding for aproduct that confers on the transformants a survival advantage, toenable their selection. Genes coding for such selectable markers includethe E. coli gpt gene which confers resistance to mycophenolic acid, theneo gene from transposon Tn5 which confers resistance to the antibioticG418 and to neomycin, the dhfr sequence from murine cells or E. coliwhich changes the phenotype of DHFR− cells into DHFR+ cells, and the tkgene of herpes simplex virus, which makes TK− cells phenotypically TK+cells. Both transient expression and stable expression can providetransformed cell lines, and membrane preparations derived therefrom, foruse in ligand screening assays.

For use in screening assays, cells transiently expressing thereceptor-encoding DNA can be stored frozen for later use, but becausethe rapid rate of plasmid replication will lead ultimately to celldeath, usually in a few days, the transformed cells should be used assoon as possible. Such assays may be performed either with intact cells,or with membrane preparations derived from such cells. The membranepreparations typically provide a more convenient substrate for theligand binding experiments, and are therefore preferred as bindingsubstrates. To prepare membrane preparations for screening purposes,i.e., ligand binding experiments, frozen intact cells are homogenizedwhile in cold water suspension and a membrane pellet is collected aftercentrifugation. The pellet is then washed in cold water, and dialyzed toremove endogenous EAA ligands such as glutamate, that would otherwisecompete for binding in the assays. The dialyzed membranes may then beused as such, or after storage in lyophilized form, in the ligandbinding assays. Alternatively, intact, fresh cells harvested about twodays after transient transfection or after about the same periodfollowing fresh plating of stably transfected cells, can be used forligand binding assays by the same methods as used for membranepreparations. When cells are used, the cells must be harvested by moregentle centrifugation so as not to damage them, and all washing must bedone in a buffered medium, for example in phosphate-buffered saline, toavoid osmotic shock and rupture of the cells.

The binding of a substance, i.e., a candidate ligand, to a human GluR3receptor of the invention is evaluated typically using a predeterminedamount of cell-derived membrane (measured for example by proteindetermination), generally from about 25 ug to 100 ug. Generally,competitive binding assays will be useful to evaluate the affinity of atest compound relative to AMPA. This competitive binding assay can beperformed by incubating the membrane preparation with radiolabelledAMPA, for example [3H]-AMPA, in the presence of unlabelled test compoundadded at varying concentrations. Following incubation, either displacedor bound radiolabelled AMPA can be recovered and measured, to determinethe relative binding affinities of the test compound and AMPA for theparticular receptor used as substrate. In this way, the affinities ofvarious compounds for the AMPA-binding human EAA receptors can bemeasured. Alternatively, a radiolabelled analogue of glutamate may beemployed in place of radiolabelled AMPA, as competing ligand.

As an alternative to using cells that express receptor-encoding DNA,ligand characterization may also be performed using cells for exampleXenopus oocytes, that yield functional membrane-bound receptor followingintroduction by injection either of receptor-encoding messenger RNA intothe oocyte cytoplasm, or of receptor-encoding DNA into the oocytenucleus. To generate the messenger RNA of cytoplasmic delivery, thereceptor-encoding DNA is typically subcloned first into a plasmidicvector adjacent a suitable promoter region, such as the T3 or T7bacteriophage promoters, to enable transcription into RNA message. RNAis then transcribed from the inserted gene in vitro, collected and theninjected into Xenopus oocytes. Following the injection of nL volumes ofan RNA solution, the oocytes are left to incubate for up to severaldays, and are then tested for the ability to respond to a particularligand molecule supplied in a bathing solution. Since functional EAAreceptors act in part by operating a membrane channel through which ionsmay selectively pass, the functioning of the receptor in response to aparticular ligand molecule in the bathing solution may typically bemeasured as an electrical current utilizing microelectrodes insertedinto the cell, in the established manner.

In addition to using the receptor-encoding DNA to construct cell linesuseful for ligand screening, expression of the DNA can, according toanother aspect of the invention, be performed to produce fragments ofthe receptor in soluble form, for structure investigation, to raiseantibodies and for other experimental uses. It is expected that theportion of the human GluR3 receptor responsible for AMPA-binding resideson the outside of the cell, i.e., is extracellular. It is thereforedesirable in the first instance to facilitate the characterization ofthe receptor-ligand interaction by providing this extracellularligand-binding domain in quantity and in isolated form, i.e., free fromthe remainder of the receptor. To accomplish this, the full-length humanGluR receptor-encoding DNA may be modified by site-directed mutagenesis,so as to introduce a translational stop codon into the extracellularN-terminal region, immediately before the sequence encoding the firsttransmembrane domain (TM1), i.e., before residue 527 as shown in FIGS.1A-1E SEQ ID NOS: 1)and 2. Since there will no longer be produced anytransmembrane domain(s) to “anchor” the receptor into the membrane,expression of the modified gene will result in the secretion, in solubleform, of only the extracellular ligand-binding domain. Standardligand-binding assays may then be performed to ascertain the degree ofbinding of a candidate compound to the extracellular domain so produced.It may of course be necessary, using site-directed mutagenesis, toproduce several different versions of the extracellular regions, inorder to optimize the degree of ligand binding to the isolated domains.

Alternatively, it may be desirable to produce an extracellular domain ofthe receptor which is not derived from the amino-terminus of the matureprotein, but rather from the carboxy-terminus instead, for exampledomains immediately following the fourth transmembrane domain (TM4),i.e., residing between amino acid residues 817-866 inclusive (FIGS.1A-1E SEQ ID NO: 1 and 2). In this case, site-directed mutagenesisand/or PCR-based amplification techniques may readily be used to providea defined fragment of the gene encoding the receptor domain of interest.Such a DNA sequence may be used to direct the expression of the desiredreceptor fragment, either intracellularly, or in secreted fashion,provided that the DNA encoding the gene fragment is inserted adjacent toa translation start codon provided by the expression vector, and thatthe required translation reading frame is carefully conserved.

It will be appreciated that the production of such AMPA-bindingfragments of a GluR3 receptor may be accomplished in a variety of hostcells. Mammalian cells such as CHO cells may be used for this purpose,the expression typically being driven by an expression promoter capableof high-level expression, for example the CMV (cytomegalovirus)promoter. Alternately, non-mammalian cells, such as insect Sf9(Spodoptera frugiperda) cells may be used, with the expression typicallybeing driven by expression promoters of the baculovirus, for example thestrong, late polyhedrin protein promoter. Filamentous fungal expressionsystems may also be used to secrete large quantities of suchextracellular domains of the EAA receptor. Aspergillus nidulans, forexample, with the expression being driven by the alcA promoter, wouldconstitute such an acceptable system. In addition to such expressionhosts, it will be further appreciated that any prokaryotic or othereukaryotic expression system capable of expressing heterologous genes orgene fragments, whether intracellularly or extracellularly would besimilarly acceptable.

For use particularly in detecting the presence and/or location of ahuman GluR3 receptor, for example in brain tissue, the present inventionalso provides, in another of its aspects, labelled antibody to a humanGluR3 receptor. To raise such antibodies, there may be used as immunogeneither the intact, soluble receptor or an immunogenic fragment thereofi.e. a fragment capable of eliciting an immune response, produced in amicrobial or mammalian cell host as described above or by standardpeptide synthesis techniques. Regions of human GluR3 receptorparticularly suitable for use as immunogenic fragments include thosecorresponding in sequence to an extracellular region of the receptor, ora portion of the extracellular region, such as peptides consisting ofresidues 1-526 or a fragment thereof comprising at least about 10residues, including particularly fragments containing residues 178-193or 479-522; and peptides corresponding to the region betweentransmembrane domains TM-2 and TM-3, such as a peptide consisting ofresidues 594-603. Peptides consisting of the C-terminal domain (residues817-866), or fragment thereof, may also be used for the raising ofantibodies.

The raising of antibodies to the selected human GluR3 receptor orimmunogenic fragment can be achieved, for polyclonal antibodyproduction, using immunization protocols of conventional design, and anyof a variety of mammalian hosts, such as sheep, goats and rabbits.Alternatively, for monoclonal antibody production, immunocytes such assplenocytes can be recovered from the immunized animal and fused, usinghybridoma technology, to a myeloma cells.

The fusion products are then screened by culturing in a selectionmedium, and cells producing antibody are recovered for continuousgrowth, and antibody recovery. Recovered antibody can then be coupledcovalently to a detectable label, such as a radiolabel, enzyme label,luminescent label or the like, using linker technology established forthis purpose.

In detectably labelled form, e.g. radiolabelled form, DNA or RNA codingfor a human GluR3 receptor, and selected regions thereof, may also beused, in accordance with another aspect of the present invention, ashybridization probes for example to identify sequence-related genesresident in the human or other mammalian genomes (or cDNA libraries) orto locate the human GluR3-encoding DNA in a specimen, such as braintissue. This can be done using either the intact coding region, or afragment thereof having radiolabelled e.g. ³²P, nucleotides incorporatedtherein. To identify the human GluR3-encoding DNA in a specimen, it isdesirable to use either the full length cDNA coding therefor, orfragment which is unique thereto. With reference to FIGS. 1A-1E and3A-3F, SEQ ID NOS: 1-4such nucoleotide fragments include thosecomprising at least about 17 nucleic acids, and otherwise correspondingin sequence to a region coding for an extracellular N-terminal orC-terminal region of the receptor, or representing a 5′-untranslated or3′-untranslated region thereof. Such oligonucleotide sequences, and theintact gene itself, may also be used of course to clone humanGluR3-related human genes, particularly cDNA equivalents thereof, bystandard hybridization techniques.

EXAMPLE 1 Isolation of DNA Coding for the Human GluR3A Receptor

The particular strategy used to clone the human GluR3A receptor isdepicted schematically in FIG. 2, and described in greater detail below.

cDNA coding for the human GluR3A receptor was identified by probinghuman hippocampal cDNA that was obtained as an EcoRI-based lambda phagelibrary (lambda ZAP) from Stratagene Cloning Systems (La Jolla, Calif.,U.S.A.). The cDNA library was probed initially with a 1.1 kb EcoRI/EcoRIDNA fragment constituting the 3′ region of a kainate-binding human EAAreceptor, designated humEAA 1a. This particular kainate-binding receptoris described in our co-pending U.S. patent application Ser. No.07/750,090 filed Aug. 26, 1991 and incorporated herein by reference. DNAcoding for the human EAA 1a receptor, and from which the 1.1 kb probemay be recovered, was deposited under terms of the Budapest Treaty, withthe American Type Culture Collection in Rockville, Md. U.S.A. on Aug.21, 1991 under accession number ATCC 75063.

Hybridizations using the probe were carried out at 30 C overnight, andfilters were washed with 2×SSC containing 0.5% SDS at 25 C for 5minutes, followed by a 15 minute wash at 50 C with 2×SSC containing 0.5%SDS. The final wash was with 1×SSC containing 0.5% SDS at 50 C for 15minutes. Filters were exposed to X-ray film (Kodak) overnight. Of 10⁶clones screened under the following hybridization conditions (6×SSC, 50%formamide, 5% Denhardt's solution, 0.5% SDS, 100 ug/ml denatured salmonsperm DNA), only two hippocampal cDNA library inserts were identified,one about 1.6 kb and designated RKCH521 and another about 2.2 kb anddesignated RKCH221 (FIG. 2). For sequencing, the '521 and the '221phages were plaque purified, then excised as phagemids according to thesupplier's specifications, to generate insert-carrying Bluescript-SKvariants of the phagemid vector. Sequencing of the '221 clone across itsentire sequence revealed a putative ATG initiation codon together withabout 78 bases of 5′ non-coding region and about 2.1 kb of codingregion. Sequencing across the '521 insert revealed a significant regionof overlap with the '221 insert, and provided some additional 3′sequence, although no termination codon was located.

There being no termination codon apparent in the '521 sequence, a 3′region of the gene was sought. For this purpose, there was firstsynthesized an oligonucleotide probe capable, of annealing to the 3′region of the rat GluR3 receptor sequence (SEQ ID NO: 7) reported byKeinanen et al, supra. The specific sequence of the 32-P-labelled probeis provided below:

5′-ACACTCAGAATTACGCTACATACAGAGAAGGCTACAACGT-3′

The same hippocampal cDNA library was then re-screened using therat-based probe and under the following hybridization conditions; 6×SSC,25% formamide, 5% Dernhardt's solution, 0.5% SDS, 100 ug/ml denaturedsalmon sperm DNA, 42 C. This revealed a 1.2 kb insert, designatedRKCSHG132. Sequencing of the entire insert revealed 5′ overlap with the3′ end of the previously isolated '521 insert, and also revealed atermination codon as well as about 15 bases of 3′ non-translatedsequence.

To provide the entire coding region in an intact clone, the strategyshown in FIG. 2 was employed, to generate the phagemid pBS/HumGluR3Awhich carries the hGluR3A-encoding DNA as a 2.8 kb EcoRI/EcoRI insert ina 3.0 kb Bluescript-SK phagemid background. The entire sequence of theEcoRI/EcoRI insert is provided in FIGS. 1A-1E and SEQ ID NO: 1.

The 5.8 kb phagemid pBS/humGluR3A was deposited, under the terms of theBudapest Treaty, with the American Type Culture Collection in Rockville,Md. USA on Mar. 19, 1992, and has been assigned accession number ATCC75218.

EXAMPLE 2 Isolation of DNA Coding for Human GluR3B Receptor

A human fetal brain cDNA library was also screened in the search forhuman GluR receptors. This particular library was obtained as anEcoRI-based lambda gt 10 library from Strategene Cloning Systems (LaJolla, Calif., U.S.A.). The library was first screened using ashybridization probe an oligonucleotide capable of hybridizing to a 3′region of the reported rat GluR3 gene sequence. Screening usinghybridization conditions as noted above (6×SSC, 25% formamide, 42 C,etc.) revealed one insert about 2.3 kb in size, designated RKCSFG34.After excision to release Bluescript-SK phagemids carrying the insert,sequencing revealed substantial sequence identity between the '34 insertand the 3′ end of the earlier isolated GluR3A clone, and suggested thatthe 5′ end of the gene encoded on partially on the '34 insert wasmissing. To provide an assembled gene, a 5′ region was excised from theGluR3A insert and used to generate the 5′ end of the '34 insert, at aninternal HindIII site. This was achieved as depicted schematically inFIG. 4. The resulting intact clone was designated human GluR3B.

Sequence comparison between the GluR3A clone of example 1 and the GluR3Bclone of this example revealed only a short region of dissimilaritywhich is illustrated, in terms of amino acid sequence, in FIG. 6 (thesequences are also shown in (SEQ ID NOS: 5 and 6).

The 6.1 kb phagemid pBS/humGluR3B was deposited, under the terms of theBudapest Treaty, with the American Type Culture Collection in Rockville,Md. USA on Mar. 19, 1992, and has been assigned accession number ATCC75219.

EXAMPLE 3 Construction of Genetically Engineered Cells Producing HumanGluR3 Receptor

For transient expression in mammalian cells, cDNA coding for the humanGluR3A receptor was incorporated into the mammalian expression vectorpcDNAI, which is available commercially from Invitrogen Corporation (SanDiego, Calif., USA; catalogue number V490-20). This is a multifunctional4.2 kb plasmid vector designed for cDNA expression in eukaryoticsystems, and cDNA analysis in prokaryotes. Incorporated on the vectorare the CMV promoter and enhancer, splice segment and polyadenylationsignal, an SV40 and Polyoma virus origin of replication, and M13 originto rescue single strand DNA for sequencing and mutagenesis, Sp6 and T7RNA promoters for the production of sense and anti-sense RNA transcriptsand a Col E1-like high copy plasmid origin. A polylinker is locatedappropriately downstream of the CMV promoter (and 3′ of the T7promoter).

To facilitate incorporation of the GluR3A receptor-encoding cDNA into anexpression vector, a NotI site was introduced onto the 5′ flank of theBluescript-SK cDNA insert, and the cDNA insert was then released frompBS/humGluR3A as a 2.8 kb NotI/NotI fragment, which was thenincorporated at the NotI site in the pcDNAI polylinker. Sequencingacross the NotI junction was performed, to confirm proper insertorientation in pcDNAI. The resulting plasmid, designatedpcDNAI/humGluR3A, was then introduced for transient expression into aselected mammalian cell host, in this case the monkey-derived,fibroblast like cells of the COS-1 lineage (available from the AmericanType Culture Collection, Rockville, Md. as ATCC CRL 1650).

For transient expression of the GluR3A-encoding DNA, COS-1 cells weretransfected with approximately 8 ug DNA (as pcDNA1/humGluR3A) per 10⁶COS cells, by DEAE-mediated DNA transfection and treated withchloroquine according to the procedures described by Maniatis et al,supra. Briefly, COS-1 cells were plated at a density of 5×10⁶ cells/dishand then grown for 24 hours in FBS-supplemented DMEM/F12 medium. Mediumwas then removed and cells were washed in PBS and then in medium. Therewas then applied on the cells 10 ml of a transfection solutioncontaining DEAE dextran (0.4 mg/ml), 100 uM chloroquine, 10% NuSerum,DNA (0.4 mg/ml) in DMEM/F12 medium. After incubation for 3 hours at 37C, cells were washed in PBS and medium as just described and thenshocked for 1 minute with 10% DMSO in DMEM/F12 medium. Cells wereallowed to grow for 2-3 days in 10% FBS-supplemented medium, and at theend of incubation dishes were placed on ice, washed with ice cold PBSand then removed by scraping. Cells were then harvested bycentrifugation at 1000 rpm for 10 minutes and the cellular pellet wasfrozen in liquid nitrogen, for subsequent use in ligand binding assays.Northern blot analysis of a thawed aliquot of frozen cells confirmedexpression of receptor-encoding cDNA in cells under storage.

In a like manner, stably transfected cell lines can also prepared usingtwo different cell types as host: CHO K1 and CHO Pro5. To constructthese cell lines, cDNA coding for human GluR3A was incorporated into themammalian expression vector pRC/CMV (Invitrogen), which enables stableexpression. Insertion at this site placed the cDNA under the expressioncontrol of the cytomegalovirus promoter and upstream of thepolyadenylation site and terminator of the bovine growth hormone gene,and into a vector background comprising the neomycin resistance gene(driven by the SV40 early promoter) as selectable marker.

To introduce plasmids constructed as described above, the host CHO cellsare first seeded at a density of 5×10⁵ in 10% FBS-supplemented MEMmedium. After growth for 24 hours, fresh medium are added to the platesand three hours later, the cells are transfected using the calciumphosphate-DNA co-precipitation procedure (Maniatis et al, supra).Briefly, 3 ug of DNA is mixed and incubated with buffered calciumsolution for 10 minutes at room temperature. An equal volume of bufferedphosphate solution is added and the suspension is incubated for 15minutes at room temperature. Next, the incubated suspension is appliedto the cells for 4 hours, removed and cells were shocked with mediumcontaining 15% glycerol. Three minutes later, cells are washed withmedium and incubated for 24 hours at normal growth conditions. Cellsresistant to neomycin are selected in 10% FBS-supplemented alpha-MEMmedium containing G418 (1 mg/ml). Individual colonies of G418-resistantcells are isolated about 2-3 weeks later, clonally selected and thenpropogated for assay purposes.

EXAMPLE 4 Ligand Binding Assays

Transfected cells in the frozen state were resuspended in ice-colddistilled water using a hand homogenizer, sonicated for 5 seconds, andthen centrifuged for 20 minutes at 50,000 g. The supernatant wasdiscarded and the membrane pellet stored frozen at −70 C.

COS cell membrane pellets were suspended in ice cold 50 mM Tris-HCl (pH7.55, 5 C) and centrifuged again at 50,000 g for 10 minutes in order toremove endogenous glutamate that would compete for binding. Pellets wereresuspended in ice cold 50 mM Tris-HCl (pH 7.55) buffer and theresultant membrane preparation was used as tissue source for bindingexperiments described below. Proteins were determined using the PierceReagent with BSA as standard.

Binding assays were then performed, using an amount of COS-derivedmembrane equivalent to from 25-100 ug as judged by protein determinationand selected radiolabelled ligand. In particular, for AMPA-bindingassays, incubation mixtures consisted of 25-100 ug tissue protein andD,L-alpha-[5-methyl-3H]amino-3-hydroxy-5-methylisoxazole-4-propionicacid (3H-AMPA, 27.6 Ci/mmole, 10 nM final) with 0.1M KSCN and 2.5 mMCaCl₂ in the 1 ml final volume. Non-specific binding was determined inthe presence of 1 mM L-glutamate. Samples were incubated on ice for 60minutes in plastic minivials, and bound and free ligand were separatedby centrifugation for 30 minutes at 50,000 g. Pellets were washed twicein 6 ml of the cold incubation buffer, then 5 ml of BeckmanReady-Protein Plus scintillation cocktail was added, for counting.

For kainate-binding assays, incubation mixtures consisted of 25-100 ugtissue protein and [vinylidene-3H] kainic acid (58 Ci/mmole, 5 nM final)in the cold incubation buffer, 1 ml final volume. Non-specific bindingwas determined in the presence of 1 mM L-glutamate. Samples wereincubated as for the AMPA-binding assays, and bound and free ligand wereseparated by rapid filtration using a Brandel cell harvester and GF/Bfilters pre-soaked in ice-cold 0.3% polyethyleneimine. Filters werewashed twice in 6 ml of the cold incubation buffer, then placed inscintillation vials with 5 ml of Beckman Ready-Protein Plusscintillation cocktail for counting.

Assays performed in this manner, using membrane preparations derivedfrom the human GluR3A receptor-producing COS cells, revealed specificbinding of 25-30 fmole/mg protein, at 10 nM [3H]-AMPA (FIG. 7). Mocktransfected cells exhibited no specific binding of any of the ligandstested. These results demonstrate clearly that the human GluR3 receptoris binding AMPA with specificity. This activity, coupled with the factthat there is little or no demonstrable binding of either kainate orNMDA, clearly assigns the human GluR3 receptor to be of the AMPA type ofEAA receptor. Furthermore, this binding profile indicates that thereceptor is binding in an authentic manner, and can therefore reliablypredict the ligand binding “signature” of its non-recombinantcounterpart from the human brain. These features make the recombinantreceptor especially useful for selecting and characterizing ligandcompounds which bind to the receptor, and/or for selecting andcharacterizing compounds which may act by displacing other ligands fromthe receptor. The isolation of the GluR3 receptor genes in substantiallypure form, capable of being expressed as a single, homogeneous receptorspecies, therefore frees the ligand binding assay from the lack ofprecision introduced when complex, heterogeneous receptor preparationsfrom human and other mammalian brains are used to attempt suchcharacterizations.

We claim:
 1. A method of assaying a substance for binding to humanGluR3, which comprises the steps of: incubating a cellular host, or amembrane preparation derived from said cellular host, with labelledGluR3 ligand to form a ligand/receptot complex, the cellular host havingincorporated expressibly therein a heterologous polynucleotide thatencodes a human GluR3 selected from the group consisting of human GluR3Aand human GluR3B, removing unbound ligand, and measuring the amount ofligand displaced from or remaining in the receptor/ligand complex.
 2. Amethod as claimed in claim 1, wherein the cellular host has incorporatedexpressibly therein a heterologous polynucleotide that encodes humanGluR3A having the sequence of SEQ ID NO:2.
 3. A method as claimed inclaim 1, wherein the cellular host has incorporated expressibly thereina heterologous polynucleotide that encodes human GluR3B having thesequence of SEQ ID NO:4.
 4. A method as claimed in claim 1, wherein theheterologous polynucleotide is plasmid pBS/humGluR3A (ATCC 75218).
 5. Amethod as claimed in claim 1, wherein the heterologous polynucleotide isplasmid pBS/humGluR3B (ATCC 75219).
 6. A method according to claim 1,wherein the cellular host is a mammalian cell.
 7. A method according toclaim 1, wherein the cellular host is an oocyte.